Zwitter-ion-based dipole assembly quenching-pH response activated quantum dot fluorescent probe as well as preparation method and application of zwitter-ion-based dipole assembly quenching-pH response activated quantum dot fluorescent probe

Abstract

The invention discloses a zwitter-ion-based dipole assembly quenching-pH response activated quantum dot fluorescent probe as well as a preparation method and application thereof, and relates to the field of biological imaging. The preparation method comprises the following steps: reacting sulfamide betaine with lipoic acid ester to obtain a zwitter-ion LAE-AB containing a disulfide bond; then zwitter-ions LAE-AB and quantum dots are subjected to a complex reaction through disulfide bonds to obtain modified quantum dots, finally, the modified quantum dots are self-assembled to form an assembly, and the quantum dot fluorescent probe is obtained. The fluorescent probe keeps a dipole assembly state in blood, fluorescence at the moment is remarkably weakened or even completely disappears due to an aggregation fluorescence quenching effect, after the fluorescence reaches a tumor site, protonation of the nano-carrier is triggered through weak acid response to achieve a dispersed state, the size is greatly reduced while the fluorescence is recovered, and the fluorescence intensity is reduced. In addition, due to charge transformation from negative potential to positive potential in protonation, penetration of the nano-carrier into tumor cells is promoted, and the fluorescence imaging effect is further enhanced.

Description

Technical Field

[0001] This invention relates to the field of bioimaging, specifically to a quantum dot fluorescent probe based on zwitterionic dipole assembly quenching-pH response activation, its preparation method, and its application. Background Technology

[0002] The global incidence and mortality rates of cancer continue to rise, with a significant annual increase in new cases and deaths, making it a major public health problem threatening human life and health. Effective cancer treatment heavily relies on early, accurate imaging diagnosis and intraoperative real-time guidance technology. Among these, the precise differentiation between diseased and healthy tissues during surgery is a core challenge in clinical treatment: because diseased tissues are often very similar in shape and color to surrounding normal tissues, they are difficult to identify, easily leading to residual lesions or accidental removal of healthy tissue. Real-time, dynamic optical visualization of the lesion area and its margins can ensure complete removal of malignant tissue while maximizing the protection of vital organs and normal tissue functions, directly impacting postoperative survival rates, recurrence rates, and quality of life. Therefore, the development of highly specific, high signal-to-noise ratio tumor imaging technologies is an urgent clinical need.

[0003] Optical fluorescence imaging, with its outstanding advantages of low cost, no ionizing radiation, high sensitivity, and ultra-high spatiotemporal resolution, can clearly present the molecular structure and dynamic physiological processes within organisms, making it a preferred solution for in vivo imaging and intraoperative guidance. It also demonstrates irreplaceable value in early tumor diagnosis and precision surgery. Semiconductor nanoparticles, also known as quantum dots, are a class of semiconductor nanocrystals with unique photoelectric properties and are core candidate materials for optical fluorescence imaging. They possess excellent properties such as a broad absorption spectrum that can excite multiple quantum dots from a single excitation source, a sharp fluorescence peak, a narrow emission spectrum with strong anti-interference capabilities, high fluorescence intensity, good chemical stability, resistance to photobleaching, high fluorescence quantum yield, and large specific surface area. Furthermore, their optical properties can be precisely adjusted by controlling particle size and composition. These characteristics make quantum dots promising for applications in bioimaging, disease diagnosis, single-molecule detection, intracellular tracking, and drug delivery, and they are considered core carrier materials for next-generation high-performance fluorescent probes.

[0004] The main problem with quantum dots in the field of bioimaging is their low signal-to-noise ratio. They lack tissue-specific luminescence capabilities and release fluorescence signals indiscriminately in both normal and tumor tissues, causing interference between their fluorescence backgrounds and making it difficult to distinguish them accurately, which greatly limits their clinical translational applications.

[0005] Most existing quantum dot fluorescent probes are in a permanent online mode with continuous fluorescence activation, maintaining strong fluorescence output even in the normal tissue microenvironment, resulting in a high background noise level in imaging. At the same time, quantum dots are prone to non-specific aggregation due to intracellular protein adsorption and fluctuations in the physicochemical conditions of body fluids. Although this can trigger aggregation-induced fluorescence quenching (ACQ effect), this aggregation is not specific to the tumor microenvironment and cannot achieve a precise switch from low background signal in normal tissue to high target signal in tumor tissue. The target signal is masked by background noise, and the signal-to-noise ratio is insufficient to meet the precise requirements of clinical diagnosis and intraoperative guidance.

[0006] Furthermore, existing probes cannot dynamically adjust their size and charge to adapt to different environments. In tumor-targeted imaging, large-size, electrically neutral probes (100-200 nm) can achieve tumor enrichment through the EPR effect, but they are difficult to penetrate the tumor parenchyma, resulting in uneven signal distribution and insufficient intensity within the tumor. Small-size, positively charged probes (10-30 nm), while possessing penetration advantages, are easily cleared by the reticuloendothelial system and cannot effectively accumulate at the tumor site, resulting in weak target signals and low signal enrichment efficiency in the tumor region, exacerbating the signal-to-noise ratio imbalance. In addition, some responsive probes rely on chemical bond breaking to activate signals, resulting in delayed and irreversible responses. They cannot capture real-time dynamic changes in the tumor microenvironment, and their response thresholds have low matching degree with the weakly acidic environment of the tumor extracellular pH 6.0-7.0, making them prone to false activation in normal tissues, generating false positive signals. The target signal is interfered with by stray signals, leading to a significant decrease in the effective signal-to-noise ratio. Summary of the Invention

[0007] To improve the signal-to-noise ratio of fluorescent probes, this invention provides a quantum dot fluorescent probe based on zwitterionic dipole assembly quenching-pH response activation, its preparation method, and its application.

[0008] To achieve the above objectives, the specific solution adopted in this invention is as follows: a method for preparing a quantum dot fluorescent probe based on dipole assembly quenching-pH response activation of zwitterions, wherein sulfonamide betaine is reacted with thioclate to obtain zwitterions LAE-AB containing disulfide bonds, and then the zwitterions LAE-AB are complexed with quantum dots through disulfide bonds to obtain modified quantum dots, and finally the modified quantum dots are self-assembled to form an assembly, thereby obtaining the quantum dot fluorescent probe.

[0009] As a further optimization of the above technical solution, the reaction process of sulfonamide betaine and thiocate is as follows: sulfonamide betaine is dissolved in a polar aprotic solvent, an alkaline activator is added to activate the reaction, a catalytic promoter and thiocate are added, and the reaction is carried out in the dark to obtain the zwitterionic LAE-AB containing disulfide bonds.

[0010] As a further optimization of the above technical solution, the polar aprotic solvent is N,N-dimethylformamide or dimethyl sulfoxide, the alkaline activator is potassium carbonate or sodium carbonate, the catalyst promoter is potassium iodide or sodium iodide, the reaction temperature is 30-50℃ in the dark, and the reaction time is 3-4 days.

[0011] As a further optimization of the above technical solution, the process of complexing zwitterionic LAE-AB with quantum dots through disulfide bonds is as follows: zwitterionic LAE-AB is dissolved in a polar aprotic solvent, a reducing agent and quantum dot solution are added under inert gas protection, and the reaction is carried out in the dark to obtain modified quantum dots.

[0012] As a further optimization of the above technical solution, the polar aprotic solvent is N,N-dimethylformamide or dimethyl sulfoxide, the reducing agent is sodium borohydride or potassium borohydride, the reaction temperature is 25℃-40℃ in the dark, and the reaction time is 1-2 days.

[0013] As a further optimization of the above technical solution, the process of modified quantum dots self-assembling to form an assembly is as follows: the modified quantum dots are dissolved in dimethyl sulfoxide, stirred, then dialyzed overnight in a phosphate buffer solution at pH 7.4, and then freeze-dried to obtain the quantum dot fluorescent probe.

[0014] As a further optimization of the above technical solution, the preparation method of sulfonamide betaine is as follows: 1-hydroxybenzotriazole, 4-dimethylamino, carbodiimide hydrochloride and tetrachlorobutyric acid are dissolved in redistilled dichloromethane, and the reaction is activated by stirring in an ice-water bath. Then, 4-hydroxybenzenesulfonamide and triethylamine are added, and the reaction is stirred at room temperature. The product PrCl is obtained by extraction and separation purification. Then, PrCl is added to an excess of trimethylamine aqueous solution, and the reaction is stirred in an oil bath until the precipitate disappears. The sulfonamide betaine is obtained by rotation and freeze-drying.

[0015] As a further optimization of the above technical solution, the preparation process of thioctic ester is as follows: thioctic acid, 4-dimethylpyridine and N,N'-dicyclohexylcarbodiimide are dissolved in dichloromethane, activated in a nitrogen atmosphere for 30 min to 1 h, then bromotetraethylene glycol is added, and the reaction is carried out in a nitrogen atmosphere in the dark for 3-4 days. The thioctic ester is obtained by extraction and separation purification.

[0016] A quantum dot fluorescent probe prepared by the method described above.

[0017] Such as the application of the quantum dot fluorescent probes mentioned above in the preparation of drugs for the diagnosis and / or treatment of tumors.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention proposes a quantum dot fluorescent probe based on zwitterionic dipole assembly quenching-pH response activation and its preparation method. The fluorescent probe maintains a dipole assembly state in blood. Due to the aggregation fluorescence quenching effect, the fluorescence is significantly weakened or even completely disappears. When it reaches the tumor site, the nanocarrier is protonated and dispersed through a weak acidic response, resulting in fluorescence recovery and a significant reduction in size. Furthermore, the charge transition from negative to positive potential during protonation promotes the penetration of the nanocarrier into tumor cells, further enhancing the fluorescence imaging effect. Compared with existing traditional imaging methods such as magnetic resonance imaging, planar X-ray imaging, computed tomography, positron emission tomography, and single-photon emission computed tomography, the product of this invention significantly improves the signal-to-noise ratio in specific imaging and can effectively distinguish between normal tissue and tumor lesions.

[0019] 2. This invention uses zwitterionic surface functionalization to prepare quantum dot fluorescent probes, which are then self-assembled in organic solvents through dipole-dipole interactions to form large-size stable assemblies of 80-200 nm. These assemblies maintain their assembled state under normal physiological conditions and achieve efficient fluorescence quenching (i.e., offline state) through the ACQ effect, thereby reducing background fluorescence noise in normal tissues from the source. At the same time, the surface of the assembly has a stable negative potential, which reduces non-specific protein adsorption and uptake by normal tissues, prolongs the blood circulation half-life to more than 3 hours, and increases the probe enrichment at tumor sites, laying the foundation for high signal output.

[0020] Under the weakly acidic conditions of the tumor microenvironment (pH 6.0-7.0), the AB zwitterionic sulfonamide groups are rapidly protonated, triggering rapid dissociation of the assembly via electrostatic repulsion, with a response time of <10 minutes. This results in the formation of small, monodisperse quantum dots of 10-30 nm in size, simultaneously achieving efficient fluorescence recovery (i.e., online state), size reduction, and charge reversal from negative to positive. This ensures strong output of the target signal in the tumor region while enhancing the probe's penetration and endocytosis efficiency in the tumor parenchyma, further amplifying the target signal intensity. The protonation process using this fluorescent probe is a purely physical reaction, requiring no waiting for chemical bond breakage and structural rearrangement. Upon contact with an acidic environment, charge conversion and assembly dissociation are initiated immediately, with a short and reversible response time. Fluorescence regulation through aggregation quenching and dispersion recovery achieves tumor-environment-specific activation, improving the signal-to-noise ratio of fluorescence imaging.

[0021] 3. This invention relies on the rapid and reversible physical reaction characteristics of protonation of zwitterionic AB to achieve a specific response to the tumor microenvironment, avoid false positive signals caused by the misactivation of normal tissue, ensure accurate switching between low background noise and high target signal, and ultimately significantly improve the signal-to-noise ratio of tumor bioimaging. It provides high-contrast optical probe support for early accurate diagnosis of tumors and real-time boundary assessment during surgery, and helps optimize clinical treatment effects. Attached Figure Description

[0022] Figure 1 The 1H NMR spectra of LAE and LAE-AB are shown. Figure 2 The image shows the 1H NMR spectrum of the fluorescent probe LAQJ. Figure 3 Infrared spectra of LAE and LAE-AB; Figure 4 The infrared spectrum of the fluorescent probe LAQJ is shown below. Figure 5 The intensity of the fluorescence emission spectra of the modified quantum dots and the assembled fluorescent probe LAQJ at different pH values; Figure 6 The particle size of the fluorescent probe LAQJ after assembly at different pH values; Figure 7 Transmission electron microscopy images of LAQ and LAQJ at different pH values; Figure 8 The permeability of LAQJ in in vitro cells; Figure 9 The in vivo fluorescence imaging effect of LAQJ; Figure 10 This is a diagram showing the half-life of the fluorescent probe LAQJ in blood circulation. Figure 11 A schematic diagram of the synthesis pathways for LAE and LAE-AB; Figure 12 This is a schematic diagram illustrating the working principle of the fluorescent probe LAQJ. Detailed Implementation

[0023] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. Parts not described or disclosed in detail in the following embodiments of the present invention should be understood as prior art known or should be known by those skilled in the art.

[0024] This invention discloses a method for preparing a quantum dot fluorescent probe based on zwitterionic dipole assembly quenching-pH response activation. The method involves reacting sulfonamide betaine with thioclate to obtain a zwitterionic ion LAE-AB containing disulfide bonds, then complexing the zwitterionic ion LAE-AB with quantum dots through disulfide bonds to obtain modified quantum dots, and finally self-assembling the modified quantum dots to form an assembly, thereby obtaining the quantum dot fluorescent probe.

[0025] Specifically, it includes the following steps: 1) Preparation of thioclate Lipoic acid, 4-dimethylpyridine (DMAP), and N,N'-dicyclohexylcarbodiimide (DCC) were dissolved in 60 ml of dichloromethane and activated under nitrogen for 30 min to 1 h. Then, bromotetraethylene glycol was added and the mixture was reacted under nitrogen at 30 °C in the dark for 3 to 4 days. The product, lipoic acid ester, abbreviated as LAE, was purified by extraction and separation.

[0026] 2) Preparation of sulfonamide betaine 1-Hydroxybenzotriazole (HOBt), 4-dimethylamino (DMAP), carbodiimide hydrochloride (EDC·HCl), and tetrachlorobutyric acid were added to a reaction flask and dissolved in redistilled dichloromethane. The reaction was activated by stirring in an ice-water bath for 1 hour. Then, 4-hydroxybenzenesulfonamide and triethylamine were added, and the reaction was stirred at room temperature for 4 days. The product was purified by extraction and separation, and the product was designated PrCl. PrCl was then added to an excess of trimethylamine aqueous solution, and the reaction was stirred in an oil bath at 70°C for several days until the precipitate disappeared. The product was then removed by rotary evaporation and freeze-drying to remove excess triethylamine and solvent, yielding sulfonamide betaine, abbreviated as AB.

[0027] 3) Preparation of LAE-AB Sulfoamide betaine was dissolved in a polar aprotic solvent, and the reaction was activated by adding a basic activator. The hydroxyl groups were activated for 30 min–1 h. Then, a catalyst promoter and thioclate ester (LAE) were added, and the reaction was carried out in the dark to obtain the zwitterionic LAE-AB containing disulfide bonds. The polar aprotic solvent was N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO). The polar aprotic solvent does not form hydrogen bonds with phenoxy anions, which can significantly improve the nucleophilic activity, and both thioclate ester and sulfoamide betaine maintain good stability in this solvent. The basic activator was potassium carbonate (K2CO3) or sodium carbonate. The role of the basic activator is to convert the phenolic hydroxyl group into phenoxy anion, thereby enhancing its nucleophilicity. The catalyst promoter was potassium iodide (KI) or sodium iodide. The catalyst promoter is a halide exchange catalyst, which generates iodoalkane with higher reactivity. The reaction was carried out in the dark at a temperature of 30–50 °C for 3–4 days, with stirring during the reaction.

[0028] 4) Preparation of modified quantum dots A zwitterionic LAE-AB solution was prepared by dissolving the zwitterionic LAE-AB in a polar aprotic solvent. The LAE-AB solution was then diluted in deionized water, and a reducing agent and quantum dot solution were added under nitrogen atmosphere. The reaction was carried out in the dark to obtain modified quantum dots, abbreviated as LAQ. The polar aprotic solvent was N,N-dimethylformamide or dimethyl sulfoxide, the reducing agent was sodium borohydride or potassium borohydride, the reaction temperature was 25℃-40℃ (preferably room temperature), and the reaction time was 1-2 days.

[0029] 5) Preparation of quantum dot fluorescent probes The modified quantum dots were dissolved in dimethyl sulfoxide at room temperature to obtain a modified quantum dot dispersion. The dispersion was stirred vigorously and then dialyzed overnight in a phosphate buffer solution at pH 7.4. The dispersion was then lyophilized to obtain the quantum dot fluorescent probe, abbreviated as LAQJ.

[0030] The present invention also discloses the use of quantum dot fluorescent probes in the preparation of drugs for the diagnosis and / or treatment of tumors.

[0031] This invention combines sulfonamide betaine zwitterion (AB) with thioclate (LAE) to form LAE-AB, which is then modified into quantum dots. This surpasses the conventional pH-responsive characteristics of sulfonamide betaine zwitterion (AB) alone, achieving dynamic dual regulation of size and charge. While AB alone can only achieve pH-responsive fluorescence switching, LAE-AB-modified quantum dots allow the probe to self-assemble into large assemblies of 80-200 nm in a normal physiological environment (pH 7.4) via dipole-dipole interactions. These assemblies efficiently enrich tumor cells through the EPR effect. In the acidic environment of the tumor (pH 6.0-7.0), they rapidly dissociate into 10-30 nm monodisperse quantum dots, simultaneously undergoing a charge reversal from negative to positive, thus resolving the functional imbalance between enrichment and penetration in traditional probes.

[0032] The brominated polyethylene glycol segment of LAE works synergistically with the zwitterionic ions of AB to form a stable negative potential on the surface of the assembly, which significantly reduces non-specific protein adsorption and normal tissue uptake, prolongs the blood circulation half-life to more than 3 hours, significantly increases the enrichment amount in tumor sites, and has better biocompatibility, avoiding the problems of easy in vivo clearance and insufficient stability that may exist with simple AB modification.

[0033] The pH response of simple AB can only trigger a change in its own charge, while LAE-AB is stably complexed with quantum dots through disulfide bonds, so that the assembly remains in an aggregated quenching state in the blood circulation (without fluorescence leakage). After reaching the tumor, it can be protonated and dissociated within 10 minutes, with a fluorescence recovery efficiency of over 90%. Moreover, protonation is a purely physical reaction with a rapid response, which can monitor the dynamic changes of the tumor microenvironment in real time, and the fluorescence switching response is more precise and faster.

[0034] After dissociation, the positive potential of the quantum dots reverses, generating electrostatic attraction with the negative potential on the surface of tumor cells. This significantly enhances the deep penetration ability of the tumor parenchyma and the efficiency of endocytosis, resulting in a more uniform and stronger distribution of fluorescence imaging signals within the tumor tissue.

[0035] This invention induces fluorescence quenching through aggregation. When quantum dots or their surface-modified luminescent particles aggregate, intermolecular π-π stacking, charge transfer, and hydrophobic interactions are significantly enhanced. This leads to the loss of more excited-state energy through non-radiative pathways such as vibrational relaxation and thermal dissipation, ultimately resulting in fluorescence quenching. In a dispersed state, the quantum dots can release fluorescence normally. By utilizing the ACQ effect, quantum dots aggregate in a normal physiological environment, achieving fluorescence quenching and reducing background noise. In the tumor microenvironment, they remain dispersed, releasing fluorescence normally and enhancing the target signal, thereby significantly improving the imaging signal-to-noise ratio.

[0036] Zwitterionic nanocarriers, due to the equal number of positive and negative charges in their repeating units, are electrically neutral overall, exhibiting both good biocompatibility and environmental responsiveness. Zwitterions possess unique intermolecular dipole-dipole interactions, leading to intermolecular aggregation and cross-linking. This invention prepares zwitterionic-functionalized quantum dots of sulfonamide betaine (AB). In organic solvent systems, these quantum dots mediate self-assembly to form stable assemblies rich in zwitterions. These assemblies maintain an aggregated state in normal physiological environments such as blood circulation, thereby inducing fluorescence quenching. However, under the extracellular acidic conditions (pH 6.0-7.0) of the tumor microenvironment, the sulfonamide groups rapidly protonate, disrupting the charge balance of the zwitterionic molecules. The assemblies quickly dissociate into monodisperse quantum dots due to internal electrostatic repulsion, emitting bright fluorescence, thus effectively improving the signal-to-noise ratio in imaging of normal tissues and tumors.

[0037] Example 1 A method for preparing a quantum dot fluorescent probe based on zwitterionic dipole assembly quenching-pH response activation, such as... Figure 11 As shown, it includes the following steps: 1) Preparation of thioclate 1.93 g of lipoic acid, 0.19 g of 4-dimethylpyridine (DMAP) and 1.93 g of N,N'-dicyclohexylcarbodiimide (DCC) were dissolved in 60 ml of dichloromethane. The mixture was first activated under nitrogen for 1 h, and then 2 g of bromotetraethylene glycol was added. The mixture was reacted under nitrogen at 30 °C in the dark for 4 days. The product, lipoic acid ester (LAE), was purified by extraction and separation.

[0038] 2) Preparation of sulfonamide betaine 0.844 g of 1-hydroxybenzotriazole (HOBt), 1.32 g of 4-dimethylamino (DMAP), 1.89 g of carbodiimide hydrochloride (EDC·HCl), and 0.766 g of tetrachlorobutyric acid were added to a reaction flask and dissolved in 60 ml of redistilled dichloromethane. The reaction was activated by stirring in an ice-water bath for 1 h. Then, 1.08 g of 4-hydroxybenzenesulfonamide and 1.8 ml of triethylamine were added, and the reaction was stirred at room temperature for 4 days. The product PrCl was purified by extraction and separation. PrCl was then added to an excess of trimethylamine aqueous solution, and the reaction was stirred in a 70 °C oil bath for several days until the precipitate disappeared. The product sulfonamide betaine AB was obtained by rotation and lyophilization.

[0039] 3) Preparation of LAE-AB 0.144 g AB was dissolved in 2 ml DMF, and 0.088 g K2CO3 was added to activate the hydroxyl groups for 30 min. Then, 0.106 g KI and 0.38 g LAE were added, and the mixture was stirred at 35 °C in the dark for 4 days. Dichloromethane:methanol = 5:1 was used as the developing solvent, and the reaction was observed by running the mixture on a silica gel plate. When the AB spot disappeared, the reaction was complete. The solid impurities were removed by filtration through a 450 nm polytetrafluoroethylene membrane, and the filtrate was collected. The pure product LAE-AB molecules were collected by column chromatography (eluent was n-hexane:ethyl acetate = 3:1). The collected LAE-AB molecules were dissolved in DMF solution to prepare a 50 mg / ml LAE-AB solution.

[0040] The successful preparation of LAE and LAE-AB was verified by proton NMR spectroscopy on a UNITY-plus400 NMR spectrometer and Fourier transform infrared spectroscopy on an IRTracer-100 spectrometer. Figure 1 , 2 As shown, since the disulfide bond exists in the five-membered ring of lipoic acid, through... Figure 1 The correspondence of the positions of the five-membered ring in the hydrogen NMR spectrum can indirectly prove the existence of disulfide bonds.

[0041] 4) Preparation of modified quantum dots LAQ Take 100 μl of LAE-AB solution, dilute it in 1 ml of deionized water, purge with nitrogen gas, add 1 mg of NaBH4, react for 30 min, then slowly add 100 μl of 3 mg / ml quantum dot (QD) solution, and react at room temperature in the dark for 1 day. After the reaction, dialyze using a dialysis bag with a molecular weight cutoff of 2000, and then freeze-dry to obtain pure modified quantum dots LAQ. It should be noted that the quantum dot QD used in this embodiment is CdTe / CdS quantum dots with aqueous NAC ligand; this quantum dot QD solution can be purchased directly and is dispersed in an aqueous solution at a concentration of 3 mg / ml.

[0042] 5) Preparation of Quantum Dot Fluorescent Probe LAQJ At room temperature, 1 mg of LAQ was dissolved in 1 ml of DMSO to prepare a modified quantum dot solution (1 mg / ml), and it was vigorously stirred. At the same time, 2 ml of phosphate buffer (PB, 50 mM, pH 7.4) was added dropwise into the Schlenk tube and stirred overnight. The solution was dialyzed using a dialysis bag with a molecular cut-off of 2000, and then freeze-dried to obtain the quantum dot fluorescent probe LAQJ.

[0043] Next, the performance of the quantum dot fluorescent probe LAQJ was tested as follows: <pH Response Performance Test> To comprehensively verify the pH response performance of the quantum dot fluorescent probe LAQJ, the fluorescence intensity and particle size of the quantum dot fluorescent probe LAQJ in different pH buffer solutions were tested.

[0044] The quantum dot fluorescent probe LAQJ was configured into buffer systems with concentrations of 0.1 mg / ml at pH 7.4 and pH 6.5. The fluorescence intensity of the quantum dot fluorescent probe solution at different pH values was measured by a fluorescence spectrophotometer, and the particle size of the quantum dot fluorescent probe solution at different pH values was measured by a Zetasizer Lab particle size and zeta potential analyzer. Relevant charts were plotted using Origin software.

[0045] As Figure 5 、 6 、shown in Figure 7, as Figure 5 shown, the fluorescence signal of the dispersed quantum dots LAQ was strong. Due to the ACQ effect, the fluorescence signal of its aggregate LAQJ was greatly weakened at pH 7.4, but the fluorescence recovered at pH 6.5.

[0046] As Figure 6 shown, the particle size of the dispersed quantum dots LAQ was a dozen nanometers, and the size of its aggregate LAQJ increased significantly, approaching 100 nm in an environment of pH 7.4. However, in an environment of pH 6.5, due to the protonation of zwitterions, the aggregates were redispersed, and the particle size was close to that of the dispersed quantum dots LAQ.

[0047] Figure 7 Shown are the transmission electron microscope images of LAQ and LAQJ at different pH values, Figure 12 and the schematic diagram of the working principle of the fluorescent probe LAQJ. From Figure 7 、 12 it can be seen the aggregation and dispersion properties of LAQJ.

[0048] <Test on the Penetration Effect of Quantum Dot Fluorescent Probe LAQJ in vitro Cells> To evaluate the cellular uptake imaging effect of the quantum dot fluorescent probe LAQJ, HepG2 cells were observed using a fluorescence inverted microscope. Sterile 12-well plates were used, with 5 × 10⁶ cells seeded in each well. 4 We collected 10 HepG2 cells and cultured them in an incubator at 37 ℃ and 5% CO2 for 24 h to allow the cells to adhere fully and grow.

[0049] To simulate the slightly acidic environment of the tumor site, the original culture medium was replaced with DMEM medium with a pH of 6.5. Then, 10 μL of quantum dot fluorescent probe LAQJ solution (working concentration of quantum dots is 0.1 mg / ml) was added to each well plate and co-incubated with cells for 3 h to investigate the uptake of different quantum dots in cells.

[0050] After incubation, the culture medium in the wells was aspirated, and the cells were washed three times with PBS to remove untaken quantum dots. Then, 200 μL of 2.5% glutaraldehyde fixative was added to each well and the cells were incubated for 5 min to fix them. After fixation, the glutaraldehyde was aspirated, and the cells were washed three more times with PBS. Finally, 200 μL of DAPI staining solution was added to each well for nuclear staining to locate the nuclei. After staining, the cells were washed three times with PBS to remove unbound DAPI.

[0051] Cell fluorescence images were captured using an inverted fluorescence microscope. The uptake effect of the quantum dot fluorescent probe LAQJ in in vitro cells was evaluated by observing the distribution and intensity of intracellular fluorescence signals. The results are as follows: Figure 8 As shown.

[0052] <Imaging Assay of Quantum Dot Fluorescent Probe LAQJ in Vivo> To visually evaluate the responsiveness of the quantum dot fluorescent probe LAQJ in the in vivo tumor gradient acid environment, a biodistribution study was conducted using tumor-bearing mice. Suitable mice were selected and subjected to tumor-bearing treatment. When the tumor volume reached 50 mm², the distribution was determined. 3 At approximately 10:00 AM, 200 μL of LAQJ quantum dot solution was injected into tumor-bearing mice via the tail vein, with an equivalent dose of quantum dots of 10 mg / kg. The biodistribution of the fluorescent probe in the mice was observed using an in vivo imaging system (IVIS Lumina III, PerkinElmer). The results are as follows: Figure 9 As shown.

[0053] <Half-life test of quantum dot fluorescent probe LAQJ> The blood circulation half-life assay was performed using existing technology. Adult mice (30±2g) were used as the research subjects. 200 μL of LAQJ quantum dot solution was injected via the tail vein, with an equivalent dose of quantum dots of 10 mg / kg. Blood samples of 0.05 mL were collected from the tail vein at 0, 1, 2, 4, 6, 8, 12, and 24 hours post-injection. The plasma was centrifuged at 3500 rpm, and the supernatant was diluted with 1 mL of methanol containing 0.01 M HCl to precipitate proteins. This centrifugation was repeated twice (8000 rpm, 10 minutes). The supernatant was filtered through a 0.22 μm aqueous filter, and the Cd abundance in the solution was detected by ICP-MS. The data were processed to obtain the circulating metabolic information.

[0054] like Figure 10 As shown, the quantum dot fluorescent probe LAQJ has a half-life of more than 3 hours in blood circulation.

[0055] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a quantum dot fluorescent probe based on zwitterionic dipole assembly quenching-pH response activation, characterized in that, The zwitterionic ion LAE-AB containing disulfide bonds is obtained by reacting sulfonamide betaine with thioclate. The zwitterionic ion LAE-AB is then complexed with quantum dots through disulfide bonds to obtain modified quantum dots. Finally, the modified quantum dots are self-assembled to form an assembly, which is the quantum dot fluorescent probe.

2. The method for preparing a quantum dot fluorescent probe based on zwitterionic dipole assembly quenching-pH response activation according to claim 1, characterized in that, The reaction process of sulfonamide betaine and thiocate is as follows: sulfonamide betaine is dissolved in a polar aprotic solvent, a basic activator is added to activate the reaction, a catalyst and thiocate are added, and the reaction is carried out in the dark to obtain the zwitterionic LAE-AB containing disulfide bonds.

3. The method for preparing a quantum dot fluorescent probe based on zwitterionic dipole assembly quenching-pH response activation according to claim 2, characterized in that, The polar aprotic solvent is N,N-dimethylformamide or dimethyl sulfoxide, the basic activator is potassium carbonate or sodium carbonate, the catalyst is potassium iodide or sodium iodide, the reaction temperature is 30-50℃ in the dark, and the reaction time is 3-4 days.

4. The method for preparing a quantum dot fluorescent probe based on zwitterionic dipole assembly quenching-pH response activation according to claim 1, characterized in that, The process of complexing zwitterionic LAE-AB with quantum dots through disulfide bonds is as follows: zwitterionic LAE-AB is dissolved in a polar aprotic solvent, a reducing agent and quantum dot solution are added under inert gas protection, and the reaction is carried out in the dark to obtain modified quantum dots.

5. The method for preparing a quantum dot fluorescent probe based on zwitterionic dipole assembly quenching-pH response activation according to claim 4, characterized in that, The polar aprotic solvent is N,N-dimethylformamide or dimethyl sulfoxide, the reducing agent is sodium borohydride or potassium borohydride, the reaction temperature is 25℃-40℃ in the dark, and the reaction time is 1-2 days.

6. The method for preparing a quantum dot fluorescent probe based on zwitterionic dipole assembly quenching-pH response activation according to claim 1, characterized in that, The process of modified quantum dots self-assembling to form an assembly is as follows: the modified quantum dots are dissolved in dimethyl sulfoxide, stirred, then dialyzed overnight in a phosphate buffer solution at pH 7.4, and then lyophilized to obtain the quantum dot fluorescent probe.

7. The method for preparing a quantum dot fluorescent probe based on zwitterionic dipole assembly quenching-pH response activation according to claim 1, characterized in that, The preparation method of sulfonamide betaine is as follows: 1-hydroxybenzotriazole, 4-dimethylamino, carbodiimide hydrochloride and tetrachlorobutyric acid are dissolved in redistilled dichloromethane and the reaction is activated by stirring in an ice-water bath. Then, 4-hydroxybenzenesulfonamide and triethylamine are added and the reaction is stirred at room temperature. After extraction and separation purification, the product PrCl is obtained. Then, PrCl is added to an excess of trimethylamine aqueous solution and the reaction is stirred in an oil bath until the precipitate disappears. The sulfonamide betaine is obtained by rotary evaporation and freeze drying.

8. The method for preparing a quantum dot fluorescent probe based on zwitterionic dipole assembly quenching-pH response activation according to claim 1, characterized in that, The preparation process of thioctic ester is as follows: thioctic acid, 4-dimethylpyridine and N,N'-dicyclohexylcarbodiimide are dissolved in dichloromethane, activated in a nitrogen atmosphere for 30 min to 1 h, then bromotetraethylene glycol is added, and the reaction is carried out in a nitrogen atmosphere in the dark for 3-4 days. The thioctic ester is obtained by extraction and separation purification.

9. A quantum dot fluorescent probe prepared by the method described in any one of claims 1-8.

10. The use of the quantum dot fluorescent probe as described in claim 9 in the preparation of medicaments for the diagnosis and / or treatment of tumors.

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